University of Warwick institutional repository: http://go.warwick.ac.uk/wrap This paper is made available online in accordance with publisher policies. Please scroll down to view the document itself. Please refer to the repository record for this item and our policy information available from the repository home page for further information. To see the final version of this paper please visit the publisher’s website. Access to the published version may require a subscription. Author(s): Whipps, J.M.; Hand, P.; Pink, D.; Bending, G.D. Article Title: Phyllosphere microbiology with special reference to diversity and plant genotype Year of publication: 2008 Link to published version: http://dx.doi.org/10.1111/j.1365- 2672.2008.03906.x Publisher statement: The definitive version is available at www.blackwell-synergy.com
1 Phyllosphere microbiology with special reference to diversity and plant
2 genotype
3 John M. Whipps, Paul Hand, David Pink and Gary D. Bending
4 Warwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK
5
6 Summary
7 The phyllosphere represents the habitat provided by the aboveground parts of plants, and on a
8 global scale supports a large and complex microbial community. Microbial interactions in the
9 phyllosphere can affect the fitness of plants in natural communities, the productivity of
10 agricultural crops, and the safety of horticultural produce for human consumption. The
11 structure of phyllosphere communities reflects immigration, survival and growth of microbial
12 colonists, which is influenced by numerous environmental factors in addition to leaf physico-
13 chemical properties. The recent use of culture independent techniques has demonstrated
14 considerable previously unrecognised diversity in phyllosphere bacterial communities. Deleted: significant 15 Furthermore there is significant recent evidence that plant genotype can play a major role in
16 determining the structure of phyllosphere microbial communities. The main aims of this
17 review are (i) to discuss the diversity of phyllosphere microbial populations (ii) to consider
18 the processes by which microbes colonise the phyllosphere (iii) to address the leaf
19 characteristics and environmental factors which determine survival and growth of colonists Deleted: to 20 (iv) to discuss microbial adaptations which allow establishment in the phyllosphere habitat
21 and (v) to evaluate evidence for plant genotypic control of phyllosphere communities. Finally,
22 we suggest approaches and priority areas for future research on phyllosphere microbiology.
23
24 Keywords: phyllosphere, bacteria, fungi, diversity, culture-independent profiling, plant
25 genotype
1 1
2 Introduction
3 The aerial parts of living plants including leaves, stems, buds, flowers and fruits provide a
4 habitat for microorganisms termed the phyllosphere. Bacteria are considered to be the
5 dominant microbial inhabitants of the phyllosphere, although archaea, filamentous fungi, and
6 yeasts may also be important. These microbes can be found both as epiphytes on the plant
7 surface and as endophytes within plant tissues (Arnold, et al. 2000; Inacio et al. 2002; Lindow
8 and Brandl 2003; Stapleton and Simmons 2006). The global surface area of the phyllosphere
9 has been estimated to total over 4 x 108 km2, supporting bacterial populations in the region of
10 1026 cells (Morris and Kinkel 2002). Furthermore, recent estimates of the diversity of
11 phyllosphere bacteria in the 20 000 vascular plants inhabiting the Brazillian Atlantic forest,
12 suggests the possible occurrence of 2 to 13 million phyllosphere bacterial species in this
13 habitat alone (Lambais et al. 2006).
14 The phyllosphere represents a niche with great agricultural and environmental
15 significance. There is growing evidence for important interactions of phyllosphere microbial
16 inhabitants which may affect the fitness of natural plant populations and the quality and
17 productivity of agricultural crops. Phyllosphere bacteria can promote plant growth and both
18 suppress and stimulate the colonisation and infection of tissues by plant pathogens (Lindow
19 and Brandl 2003; Rasche et al., 2006). Similarly, fungal endophytes of leaves may deter
20 herbivores, protect against pathogens and increase drought tolerance (Arnold et al. 2003;
21 Schweitzer et al. 2006). Furthermore, interactions in the phyllosphere zone determine the
22 extent to which human pathogens are able to colonise and survive on plant tissues, an area of
23 increasing importance with the rise in cases of human disease associated with consumption of Deleted: 2006 24 fresh salad, fruit and vegetable produce (Whipps et al. 2008).
2 1 There is evidence for functional roles within the phyllosphere microbial community which
2 given the size of the habitat could have global significance. The best studied of these is
3 nitrogen fixation. Measured rates of bacterial nitrogen fixation in the phyllosphere vary
4 widely, but in the phyllosphere of trees in some tropical habitats has been reported at rates of
5 over 60 kg N ha-1, although amounts fixed in the phyllosphere of temperate trees is generally
6 considerably lower (Freiberg 1998). Furthermore, N2 fixation or the presence of N2 fixing
7 bacteria has been reported in the phyllosphere of many crop plants (e.g. Murty 1983;
8 Miyamoto et al. 2004). Other environmentally important microbial processes for which there
9 is evidence in the phyllosphere include methanol degradation (Corpe and Rheem, 1989; Van
10 Aken et al. 2004) and nitrification (Papen et al. 2002), although the rates of these process and
11 their ubiquity within the phyllosphere remains to be elucidated.
12 Most knowledge of the structure and activities of phyllosphere microbial communities has
13 been established using culture-dependant methods. However, these are recognised to
14 significantly underestimate diversity, with only 0.1-3 % of environmental bacteria considered
15 culturable (Wagner et al. 1993). Data gathered using these methods therefore relate only to
16 culturable members of the community and provide no information on the vast majority of
17 microbes present in samples. As in other areas of environmental microbiology, the recent
18 application of culture-independent methods based on the characterisation of small subunit
19 rRNA gene sequences for microbial community analysis is providing new insights into the
20 complexity of phyllosphere microbial communities and their interactions with plants and the
21 wider environment. Deleted: The main aims of this 22 In the current paper we review the extent to which the use of culture independent review Deleted: are 23 approaches has changed our understanding of the structure and diversity of phyllosphere
24 communities. A variety of plant, microbial and environmental factors control establishment of
25 microbial communities in the phyllosphere, but recently there has been recognition of the role
3 1 that plant genotype plays in selecting phyllosphere communities. The evidence for the
2 different factors which regulate the structure of phyllosphere communities is discussed with Deleted: (i) to discuss the diversity of phyllosphere 3 special reference to the role of plant genotype. Finally, we suggest approaches and priority microbial populations (ii) to consider the processes by which microbes colonise the 4 areas for future research on phyllosphere microbiology. Although much of the phyllosphere phyllosphere (iii) to address the leaf characteristics and environmental factors which 5 literature is concerned with interactions between plant and plant pathogens (bacteria and fungi determine survival and growth of colonists (iv) to discuss microbial adaptations to the phyllosphere 6 that cause diseases in plants), in the current review emphasis is placed on studies of habitat and (v) to evaluate evidence for plant genotypic control of phyllosphere 7 microorganisms that live in the phyllosphere without causing obvious damage to the plant, as communities. Deleted: Particular attention 8 absence of disease is the normal situation in nature. will be given to recent advances in understanding gained through the application of culture 9 independent methods.
10 Microbial diversity in the phyllosphere
11 The microbial communities of the phyllosphere are diverse, supporting numerous genera of
12 bacteria, filamentous fungi, yeasts, algae and in some situations protozoans and nematodes
13 (Morris et al. 2002; Lindow and Brandl 2003). Bacteria are the most numerous and diverse
14 colonists of leaves, with culturable counts ranging between 102 to 1012 cells g leaf (Thompson
15 et al. 1993; Inacio et al. 2002). Culture-based studies of sugar beet over the whole of the
16 growing season have found more than 78 bacterial species representing 37 known bacterial
17 genera (Thompson et al. 1993). Similar studies in wheat have revealed 88 bacterial species Deleted: However, r 18 representing 37 known bacterial genera (Legard et al. 1994).
19 Recent studies have demonstrated that profiling of phyllosphere communities based on Deleted: in 20 culture dependent methods is likely to be inaccurate and to underestimate diversity (Rasche et Deleted: 21 al. 2006b). In the case of the phyllosphere, use of culture independent approaches has shown
22 that although assumptions regarding the dominant inhabitants are largely correct, the diversity Deleted: ¶ 23 of phyllosphere communities is far greater than previously recognised.Analysis of 16S rDNA
24 cloned directly from leaf samples has demonstrated that proteobacteria are the dominant
25 group found on leaves (Table 1), confirming data obtained using culture-dependant methods
4 1 (e.g. Thompson et al. 1993). α and γ- proteobacteria are generally the dominant bacterial
2 inhabitants of the phyllosphere, with bacteroidetes also usually important. β-proteobacteria Deleted: s 3 and firmicutes can also form a large part of the bacterial community in some situations, with
4 acidobacteria, actinobacteria and cyanobacteria occurring infrequently (Kadivar and Stapleton
5 2003; Idris et al. 2004; Lambais et al. 2006; Rasche et al. 2006b,c).
6 In a study of phyllosphere bacterial communities in a tropical Brazillian forest, 97 % of
7 bacterial sequences were from previously undescribed species with phyllospheres of different
8 plant species supporting from 95 to 671 bacterial species (Lambais et al. 2006). The extent to
9 which such diversity occurs in other plant species is unclear. Those sequences showing 95 %
10 or less homology to known bacterial species database entries comprised 15.2 % of sequences
11 obtained from Thlapsi geosingense (Idris et al. 2004), and 7.9, 2.3, 3.5 and 1.2 % of those
12 sequences obtained from Crocus, potato, pepper and maize respectively (Kadivar and
13 Stapleton 2003; Rasche et al. 2006 a,b, Reiter and Sessitsch, 2006). However, in a study of a
14 range of temperate agricultural crop species, 5 of 17 bands cut from 16S rRNA denaturant
15 gradient gel electrophoresis gels had less than 90 % similarity to database entries, suggesting
16 that in some situations phyllospheres of crop plants may support large numbers of novel
17 bacteria (Yang et al. 2001). The number of sequences investigated in the culture independent
18 studies conducted to date has been limited, so that only dominant members of the community
19 are likely to have been detected, and the true extent of bacterial diversity in the phyllosphere
20 therefore remains to be determined.
21 Yeasts are the major epiphytic fungal group in the phyllosphere with filamentous fungi
22 largely occurring as dormant spores rather than active mycelia except on older leaves
23 (Andrews and Harris 2000; de Jager et al. 2001). Culturable yeast populations can range
24 between 10 and 1010 colony forming units g leaf (Thompson et al. 1993, Inacio et al. 2002).
25 The diversity of culturable yeasts appears to be mostly limited to the genera Cryptococcus,
5 1 Sporobolomyces and Rhodotorula, although total species number can reach over 40, with
2 multiple species of each coexisting in the phyllosphere, together with a number of other
3 genera which occur less frequently (Thompson et al. 1993; Inacio et al. 2002; Glushakova
4 and Chernov 2004).
5 Filamentous fungus population sizes can range between 102 and 108 colony forming units g
6 leaf. Cladosporium and Alternaria are usually considered the most abundant fungi found on
7 leaves, although several other genera, including Penicillium, Acremonium, Mucor and
8 Aspergillus are also found (Thompson et al. 1993; Inacio et al. 2002). Filamentous fungi
9 appear to occur ubiquitously as endophytes, the diversity of which may be substantial,
10 particularly in long lived tropical leaves. Using culture dependant approaches, over 340
11 genetically distinct taxa were recovered from individuals of 2 tropical forest understory plant
12 species at 2 sites. Furthermore, there was evidence for host preference within the endophyte
13 community (Arnold et al. 2000). Culture independent approaches have not yet been used to
14 characterise fungal diversity in the phyllosphere.
15 There are various developing technologies which show promise to significantly increase
16 throughput of analysis to provide a finer resolution of understanding about the diversity and
17 structure of phyllosphere communities and to link diversity with functioning. Culture-
18 independent analysis using phylogenetic specific primers represents a powerful method to
19 investigate the dynamics and distribution of specific bacterial groups of interest (Sessitsch et
20 al. 2002; Miyamoto et al. 2004). Additionally multiplex TRFLP, in which several phylogentic
21 groups or functional genes can be analysed at the same time provides an opportunity to
22 improve throughput of samples in a cost effective manner (Singh et al. 2006). However, these
23 techniques remain time consuming, and future developments will depend on high throughput
24 methods. Phylogenetic microarrays clearly provide a way forward, allowing the presence and
25 amount of thousands of microorganisms to be determined simultaneously, and could also be
6 1 used to detect novel members of phylogenetic groups. Similarly, functional gene arrays
2 provide a means of characterising activity of the phyllosphere community, and when used
3 with phylogenetic microarrays, for linking community structure to function (Sessitsch et al.,
4 2006).
5
6 In order to understand and predict the diversity and structure of phyllopshere communities, it
7 is necessary to understand the biological and environmental factors which control the
8 establishment and dynamics of microbial communities on the leaf surface. This is the focus of
9 the remainder of the review.
10
11 Deleted: Microbial 12 Sources of microbes colonising the phyllosphere Deleted: colonisation Deleted: of 13 The sources of microorganisms on the phyllosphere can be manifold. Epiphytic filamentous
14 fungi, yeasts and bacteria may arrive on the leaf surface through insect-, atmosphere-, seed- or
15 even animal-borne sources. Tree buds, seeds of annual plants and the debris from previous
16 crops are likely to be the most important sources for the colonization of new plants and leaves
17 as they are a major source of bacteria already adapted to the phyllosphere (Manceau and
18 Kasempour 2002).
19 Those microorganisms that show no or limited multiplication in the phyllosphere are
20 considered transient epiphytes whereas those with the capacity for multiplication in the
21 absence of wounds are known as residual epiphytes (Suslow 2002). Microbial populations can
22 vary in size among and within plant species over short periods of time (Hirano and Upper
23 1989) as well as over the growing season (e.g. Thompson et al. 1993; Legard et al. 1994;
24 Inacio et al. 2002), with few epiphytic bacteria present on leaves shortly after emergence from
25 buds or seeds, but increasing in quantity subsequently (Hirano and Upper 1993). There is a
7 1 general succession of microbial populations on leaves over the growing season with bacteria
2 dominating initially, followed by yeasts and finally filamentous fungi (Kinkel 1997).
3 The atmospheric microflora can vary in composition and concentration diurnally and
4 seasonally as well as in response to environmental events such as rainfall and high wind
5 (Kinkel 1997; Zak 2002), directly influencing the immigration of microorganisms to the
6 phyllosphere. Local vegetation, and in areas of crop production, agricultural practices such as
7 harvesting and cultivation, also influence atmospheric microbiology and colonisation of
8 nearby plants (Lindemann et al. 1982; Lacey 1996; Lighthart 1997). Immigration of
9 microorganisms to leaves from the atmosphere can take place through impaction onto the leaf
10 surface, sedimentation or rain splash as well as from contamination with soil (Venette and
11 Kennedy 1975; Lacey 1996).
12 There is increasing evidence that microorganisms on seeds or roots can become
13 endophytic in the roots, enter the vascular system and be transferred internally to the aerial
14 parts of plants where they establish as phyllosphere endophytes (Lamb et al. 1996; Wulff et
15 al. 2003). Endophytes can also arise from ingression into the internal leaf spaces following
16 colonisation by epiphytes, suggesting that epiphytes and endophytes are really part of a
17 continuum in the phyllosphere (Wilson et al. 1999; Beattie and Lindow 1999).
18 Once microorganisms have arrived in the phyllosphere they have to become established
19 and colonise the leaf to become a residual epiphyte. The pattern of distribution of
20 microorganisms on leaves is not even. The most common sites of bacterial colonisation are in
21 the epidermal cell wall junctions (Blakeman 1985; Davis and Brlansky 1991) especially in
22 protected sites in grooves along the veins (Mansvelt and Hattingh 1987; Leben 1988; Mariano
23 and McCarter 1993), at stomata (Mew and Vera Cruz 1986; Mariano and McCarter 1993),
24 and at the base of trichomes (Mew and Vera Cruz 1986; Mansvelt and Hattingh 1987;
25 Mariano and McCarter 1993). They are also found under the cuticle (Corpe and Rheem,
8 1 1989), in depressions in the cuticle (Mansvelt and Hattingh 1987), near hydrathodes (Mew et
2 al. 1984) and in specific sites that only occur on particular plants such as stomatal pits in
3 oleander and pectate hairs in olive (Surico 1993). In general, greater numbers of bacteria are
4 found on lower than upper leaf surfaces (Leben 1988; Surico 1993) possibly due to the lower
5 leaf surface having a greater density of stomata or trichomes, or a thinner cuticular layer
6 (Beattie and Lindow 1999).
7 Bacterial populations in the phyllosphere can differ in distribution over very small scales,
8 as little as 0.1 mm2 (Kinkel et al. 1995) and are often well-described by a log-normal
9 distribution (Hirano et al. 1982; Ishimaru et al. 1991) whereas yeasts and filamentous fungi
10 may be better described by a normal distribution (Kinkel et al. 1989). Microorganisms may
11 occur individually on the leaf surface but frequently, they occur as aggregates or biofilm-like
12 structures containing bacteria, (Kinkel et al. 1995; Morris et al. 1997, 1998; Jacques et al.
13 2005), yeasts (Last 1955) and filamentous fungi (Bernstein and Carroll 1977).
14 Clearly, not all microorganisms that arrive in the phyllosphere are able to colonise and
15 grow. To some extent this reflects processes of emigration through dispersal mechanisms
16 such as rain splash, wash-off, bounce-off, water movement or removal by insects (Kinkel
17 1997). Ability to survive and grow are dependent on the environmental, physicochemical and
18 genetic features of the plant and specific properties exhibited by the phyllosphere
19 microorganisms, which together determine the structure and diversity of the microbial
20 community. Evidence for such selection is supported by the findings of Miyamoto et al.
21 (2004), in which 16S rRNA-Terminal Restriction Fragment Length Polymorphism (TRFLP)
22 with Clostridia specific primers was used to show the presence of diverse Clostridia
23 populations within Miscanthus sinensis, which were shown to be distinct to those Clostridia
24 populations inhabiting soil around plants. Furthermore, since a substantial proportion of those
25 bacteria inhabiting the phyllosphere appear to be novel to this habitat there have been
9 1 suggestions that some may be unique or specialists to this habitat (Yang et al. 2001; Lambais
2 et al. 2006).
3 There are a number of areas relating to the colonisation of phyllosphere which require more
4 complete understanding. The transmission of microorganisms from roots to aerial parts of
5 plants appears to have been a neglected area of research and the importance of this
6 environmentally protected phyllosphere colonisation route needs to be elucidated. This could
7 be particularly important for soils contaminated with human pathogens.
8
9 Deleted: affecting 10 Leaf characteristics and environmental factors controlling phyllosphere microbiology
11 Following arrival of microbial cells or propagules on the leaf surface, a variety of factors
12 determine whether cells are able to colonise the leaf, and where cells become localised.
13 Establishment is determined by interaction between leaf and environmental characteristics
14 which interact to control conditions prevailing in the phyllosphere habitat. The first point of
15 contact of microbial cells immigrating to the phyllosphere is the cuticle (Beattie 2002). This
16 waxy surface, often microcrystalline in nature, serves several functions: a diffusion barrier,
17 reducing water and solute loss and aqueous pollution ingress; as a reflectant to minimise
18 temperature fluctuations; conferring water repellancy; and providing protection from
19 pathogens (Beattie 2002). The water repellancy is particularly important in preventing
20 immigration of microorganisms to the leaf surface. This is so especially on young leaves
21 where the cuticle is intact relative to older leaves because as the cuticle erodes, wettability
22 increases (Beattie 2002). In addition, cuticle-mediated limitation of nutrient loss from the leaf
23 is particularly important in supporting epiphytic microbial populations. Use of whole cell
24 bacterial biosensors for sucrose, fructose and glucose have revealed that these sugars are
25 present only in discrete localised sites on the leaf (Leveau and Lindow 2001; Miller et al.
10 1 2001). This, and recent microscopical evidence (Monier and Lindow 2005), suggests that
2 most microbial immigrants to the phyllosphere are exposed to nutrient poor environments and
3 that only few cells randomly land in zones of relatively abundant nutrients that support
4 growth. Other discrete sites of nutrient loss such as wounds or glandular trichomes (Monier
5 and Lindow 2005), or sites of nutrient enrichment including pollen (Diem 1974) or honeydew
6 (Dik et al. 1992) also provide sites for microbial growth. Other nutrients such as N-sources or
7 iron are not considered as growth limiting to microorganisms on the phyllosphere as C-
8 sources (Lindow and Brandl 2003). Interestingly, when the resurrection fern, Polypodium
9 polypodioides, is exposed to rainfall after a period of desiccation, the complex phyllosphere
10 community undergoes changes in overall structure and activity, reflecting use of labile
11 organic substrates in the form of an enrichment culture (Jackson et al. 2006). Whether this
12 occurs with other plants is unknown.
13 Plant leaves also release a wide range of volatile organic compounds into the boundary
14 layer around leaves. These can include small molecules such as CO2 and acetone, medium
15 sized molecules including terpenoids and a number of aldehydes and alcohols, as well as large
16 molecules such as long-chain hydrocarbons and sesquiterpenoids; sulphides and nitrogen-
17 containing compounds also occur. It is unclear whether these could be nutrient sources
18 directly, but there is evidence that some of these compounds can be inhibitory or toxic to
19 some fungi (Mechaber 2002). Similarly, some proteins secreted by glandular trichomes can
20 inhibit some pathogens (Shepherd et al. 2005). There are also data to suggest that plants can
21 release a number of compounds in response to damage that not only promote microbial
22 development but can selectively inhibit microbial growth as well (Dingman 2000).
23 Characteristics of the plant species themselves may also influence the microbial carrying
24 capacity of the leaf. The total number of culturable bacteria from broad leaf succulent
25 herbaceous plants such as cucumber, lettuce and bean can be significantly higher than that
11 1 from grasses or waxy broad-leaved plants such as cabbage and citrus (O’Brien and Lindow
2 1989; Lindow and Andersen 1996; Kinkel et al. 2000). Culture independent approaches have
3 demonstrated that community structure on leaves from individuals of the same species is
4 similar, but varies significantly between species (Yang et al. 2001). Lambais et al. (2006)
5 showed that just 0.5 % of bacterial species recorded in tropical tree canopies were common to
6 all tree species. Furthermore, both bacterial and fungal population size on leaves has been
7 correlated with leaf position, plant architecture and height in the canopy (Wildman and Deleted: The extent to which 8 Parkinson, 1979; Oliveira et al. 1991; Jacques et al. 1995). We would suggest that microbial Deleted: is 9 diversity and community structure are also influenced by these factors, although this remains Deleted: influenced by these factors 10 to be shown. Deleted: elucidated
11 Information is needed to characterise the arrangement and dynamics of communities to time
12 and space, especially at the landscape scale. In particular the relative importance of
13 environmental factors, location and plant species in determining the composition and
14 dynamics of phyllosphere communities needs to be addressed. Biogeographical approaches to
15 the analysis of microbial communities show potential to elucidate these fundamental
16 relationships (Ramette and Tiedje, 2007).
17
18 Microbial adaptations to the phyllosphere habitat
19 In addition to plant and environmental factors, properties of the microbial colonists Deleted: In view of the relatively stressful environmental 20 themselves determine the extent to which they are able to establish on the leaf surface. For and physicochemical features of the phyllosphere, it is clear that microorganisms capable of 21 some microorganisms this reflects their inherent ability to survive in the existing habitat growth in the phyllosphere must exhibit a number of adaptations 22 whereas others are capable of modifying the environment to ameliorate the levels of stress for this lifestyle. Deleted: present 23 they are exposed to.
24 Culture independent analyses have indicated that tolerance to UV radiation is likely to be an
25 important selection pressure for survival and growth this habitat (Kadivar and Stapleton,
12 1 2002; Stapleton and Simmons, 2006), and most isolated phyllosphere microorganisms are
2 capable of withstanding high UV radiation levels on the leaf surface (Sundin 2002). In fungi, Deleted: presence of 3 dark melanin-type pigments are thought to play a key role as protective pigments along with
4 UV-B induced hyphal-wall thickening, the latter protecting lower levels of the fungal colony
5 (Fourtouni et al. 1998; Sundin 2002). Interestingly, the most UV-B tolerant strains of bacteria
6 from the peanut phyllosphere were those that produced pink or orange pigments (Sundin and
7 Jacobs 1999) and so multiple UV-B protectant mechanisms may be exhibited by phyllosphere
8 microorganisms.
9 A low level of water availability and nutrients are key limiting factors for microbial growth
10 in the phyllosphere so epiphytes display a variety of mechanisms to overcome these
11 limitations. For example, some epiphytic Pseudomonas spp. can release surfactants that
12 increase the wettability of leaf surfaces making it easier for microorganisms to use water and
13 increasing solubilisation and diffusion of nutrients, thereby increasing substrate availability to
14 epiphytic bacteria (Bunster et al. 1989). A number of phyllosphere bacteria have recently
15 been shown to increase permeability of the cuticle enhancing water and nutrient availability in
16 the phyllosphere (Schreiber et al. 2005). Another, potentially related, mechanism to increase
17 nutrient availability may relate to the ability to produce toxins that affect ion transport across
18 plant cell plasma membranes (Quigley and Gross 1994; Hutchison et al. 1995). Plant
19 pathogenic Pseudomonas syringae pv. syringae secrete the toxin syringomycin which
20 eventually leads to cell lysis. Nevertheless, low levels are produced by non-pathogenic
21 epiphytic strains of P. syringae pv. syringae such that necrosis and disease do not occur
22 although release of plant nutrients is still stimulated (Hutchison et al. 1995). Interestingly,
23 syringomycin also acts as a surfactant providing two possible mechanisms to enhance nutrient
24 availability in the phyllosphere.
13 1 Another, perhaps more widespread mechanism, is the production and release of plant
2 growth regulators. Production of indole-3-acetic acid (IAA) is common among bacterial
3 epiphytes (Glickman et al. 1998; Brandl et al. 2001) and is associated with enhanced nutrient
4 leakage and microbial fitness (Brandl and Lindow 1998; Manulis et al. 1998). Lindow and
5 Brandl (2003) have also made the suggestion that presence of a functional type III secretion
6 pathway in Pseudomonas fluorescens and Pseudomonas putida (Preston et al. 2001), which
7 provides the capacity to modify the local habitat, may be needed for growth and survival in
8 the phyllosphere. Production of pili and flagellae may also be important in allowing bacterial
9 attachment and colonisation of the phyllosphere (Romantschuk et al. 2002). A whole range of
10 genes and gene products that are important for phyllosphere colonisation are now being
11 identified using molecular techniques (Gal et al. 2003; Gourion et al. 2006) and may provide
12 further insights into mechanisms involved in epiphytic growth.
13 As mentioned earlier, bacterial distribution on the leaf surface is not uniform and frequently,
14 aggregates of cells occur (Morris and Monier 2003). The presence of these aggregates may
15 provide the epiphytes with an ability to colonise and survive in the phyllosphere and modify
16 the local environment. The production of extracellular polysaccharides (EPS) by bacteria may
17 protect the bacteria from water stress and help anchor the cells to the leaf surface (Morris et
18 al. 1997; Gal et al. 2003). By analogy with biofilms (Morris et al. 2002; Morris and Monier
19 2003), these aggregates may also protect from UVR, predation and bacteriocides, moderate
20 pH and gas exchange, enhance genetic exchange particularly through plasmid transfer, and
21 allow cell density – dependent behaviour. The latter, often mediated by accumulation of
22 diffusible molecules such as N- acyl homoserine lactones through quorum sensing may have
23 numerous effects on microbial behaviour including EPS and antibiotic production as well as
24 pathogenicity traits (Swift et al. 1994; Greenberg 1997). Interestingly, if signalling controls
25 the formation and functioning of aggregates, it may be possible in future to manipulate the
14 1 microbial populations on the phyllosphere if the molecular signals and receptors essential for
2 aggregate behaviour can be identified (Morris et al. 2002). Deleted: ¶ 3 Deleted: Microbe-microbe interactions in the phyllosphere¶ 4 The occurrence of large populations of microorganisms on the phyllosphere allows the 5 Plant genotype and phyllosphere microbiology possibility of extensive microbe- microbe interactions providing cells are in relatively close 6 It is clear that microbial populations in the phyllosphere can vary markedly in size and proximity. Besides the studies of aggregates already mentioned, there have been numerous 7 composition both spatially and temporally on the same plant, differ between different plants investigations of other microbial interactions, largely concerned with biological control. For 8 and parts of plants in the same place, and even differ on the same plant species in different example, some bacteria on the phyllosphere exhibit ice nucleation activity (Ice+ bacteria), 9 places (Lindemann et al. 1984; Morris and Lucotte 1993; Lindow and Andersen 1996; Kinkel and when in high numbers, these bacteria reduce the ability of the plant cells to supercool and avoid 10 1997). Much of the variability must reflect the environmental conditions prevailing at the time ice formation (Lindow 1995; Hirano and Upper 2000). Consequently, pre-emptive 11 and place of sampling, thereby influencing the processes of microbial immigration, applications of Ice- bacteria when Ice+ bacteria were at low levels resulted in effective control of the 12 emigration, growth and death. However, the microbial population that does develop must problem, apparently acting largely through competition for carbon compounds as antibiotic 13 relate to a large extent to the phenotypic characteristics exhibited by the plant, controlled production appears rare in bacterial interactions in the phyllosphere in general (Lindow 14 ultimately by its genetic make-up. Certainly, there are “hot-spots” of microbial growth on the 1988). Another successful case of bacterial biocontrol involves the use of non-pathogenic strains of 15 leaf associated with specific sites and it would be expected that these would similarly be P. fluorescens and Pantoea agglomerans to control the fireblight pathogen, Erwinia 16 under the influence of plant genetic characteristics. We suggest that within plant species, amylovora on flowers of apple and pear (Lindow and Leveau 2002). Again, pre-emptive 17 genotype has a key role in determining colonisation and establishment of microbial colonisation, in this case of the stigma, by the non-pathogenic strains lead to competitive 18 communities in the phyllosphere. However, few studies have addressed the relationship exclusion of the pathogen and disease control although antibiotic production may have been 19 between plant genetic control of phenotypic characteristics and their concomitant effects on involved to some extent in this system (Stockwell et al. 2002). ¶ Fungal-fungal interactions on the 20 microbial populations in the phyllosphere, despite its potential importance. phyllosphere can also provide disease control. For instance, on onion leaves, Gliocladium 21 Several studies have used culture-dependent approaches to investigate the impact of plant catenulatum and Aureobasidium pullulans appeared to control Botrytis aclada by antibiotic 22 genotype on phyllosphere microbiology. Adams and Kloepper (2002) showed that endophytic production whereas Ulocladium atrum acted through competitive exclusion (Köhl et al. 1997). The 23 bacteria population sizes and structure differed between 9 cotton cultivars, and in pea 5 of 11 other most widely reported mode of action involves parasitism of fungal pathogens by other fungi 24 cultivars were found to contain endophytic bacteria with one showing a higher colonisation but often this mode of action is accompanied by antibiotic production as well (Bélanger and 25 level than the others (Elvira-Recuendo and van Vuurde 2000). In a gnotobiotic system with Avis 2002) indicating that multiple modes of action for biocontrol are likely to occur in the phyllosphere overall.¶
15 1 tomato, one cultivar out of four supported fewer Pseudomonas sp. on the shoot exterior
2 following application of the bacterium to the seed (Pillay and Nowak 1997). Differences in
3 ability to support populations of Pseudomonas syringae pv. syringae were also found between
4 different cultivars of snap bean (Hirano et al. 1996; Upper et al. 2003). However, no
5 differences in occurrence of native, epiphytic mycoparasites were observed between the 3
6 main coffee cultivars or between clones of the same group (ten Hoopen et al. 2003).
7 Similarly, no differences were found between epiphytes on three cultivars of apple (Becker
8 and Manning 1983) or in endophytes in three cultivars of wheat (Larran et al. 2002).
9 Culture-independent community profiling approaches have been particularly valuable for
10 elucidating interactions between plant genotype and phyllosphere microbial community
11 structure. Several studies have indicated that different cultivars of the same plant species
12 exhibit different phyllosphere microbial populations. Phyllosphere populations of bacteria
13 were found to differ between cultivars of sweet pepper and tomato (Correa et al. 2007;
14 Rasche et al. 2006b) and both endophytes and epiphytes differed between varieties of potato
15 (Sessitch et al. 2002; Rasche et al. 2006a, c). Plant genotype differences may affect some
16 microbial communities more than others. For example, phyllosphere bacterial community
17 structure was shown to vary between wheat cultivars, although there was found to be no
18 difference in archaeal communities (Stapleton and Simmons 2006). Recently, lettuce cultivar Deleted: have no e 19 was shown to affect colonisation of leaves by Salmonella enterica serovars (Klerks et al., Deleted: on
20 2007), with significant serovar-cultivar interactions demonstrated. Furthermore, diversity of
21 endophyte bacterial populations varied between the three lettuce cultivars used, and data
22 suggested that the degree to which Salmonella enterica serovars were able to colonise plants
23 endophytically was in part determined by competitive interactions with the natural endophyte Deleted: although it was not clear whether there were 24 bacterial community. differences in overall bacterial community structure between the five lettuce cultivars investigated.
16 1 Culture dependent analysis showed that genetic modification of potato with an antibacterial
2 peptide, magainin, failed to influence the number or structure of phyllosphere bacterial or
3 fungal populations even though maganin-expressing potato tubers did exhibit lower total
4 numbers of bacteria than unmodified plants (O’Callaghan et al. 2004). In contrast, genetic
5 modification of potato with a gene producing anti-bacterial T4-lysozyme or attacin/cecropin,
6 was shown to induce greater difference in phyllosphere microbial community structure to the Deleted: 3 7 parent line, relative to variations between three cultivars (Rasche et al. 2006a, c). However,
8 field site and plant growth stage had greater effects on bacterial community structure than
9 either cultivar or genetic modification.
10 Furthermore, microbial communities selected by different genotypes can show differing
11 responses to environmental variables. Rasche et al. (2006b) showed that chilling sweet pepper
12 plants altered endophyte bacterial community structure, with the extent of the effect differing
13 between cultivars, and dependant on cultivar chilling tolerance. Similarly, in a study of wheat
14 cultivars, it was shown that the response of phyllosphere bacterial communities to UV-B
15 radiation depended on host genotype. However it was not clear whether these differences
16 reflected direct effects on the bacterial community or indirect effects associated with
17 differences in the plant responses to UV-B (Stapleton and Simmons 2006). Furthermore, plant
18 genotype can influence colonisation and survival of microbial inoculants in the phyllosphere.
19 Correa et al. (2007) showed that the survival of a plant growth promoting Azospirillum
20 inoculant differed in the phyllosphere of contrasting tomato genotypes, and that the response
21 of the phyllosphere bacterial community to inoculation varied between genotypes.
22 In the case of fungi, there is limited data on plant genotype-diversity relationships, although
23 several studies have demonstrated differences in the nature of endophytes associated with
24 contrasting host genotypes. For example, distinct communities of endophytes were shown to
17 1 be associated with different Populus hybrids, with the percentage condensed tannins in bark
2 implicated in directing these differences (Schweitzer et al. 2006).
3 Although plant genotype appears to be an important factor determining the structure of
4 phyllosphere microbial communities, the mechanisms controlling these interactions remain to
5 be elucidated. Various plant science resources are available which show potential for
6 examining plant genotype-phyllosphere microbiology interactions. In particular recombinant
7 inbred mapping populations (Asins, 2002) have the potential to identify plant genes
8 controlling leaf microbiology.
9
10 Conclusions and future directions
11 Although culture-independent molecular analysis of microbial populations in the
12 phyllosphere is still in its infancy, it is clear from recent studies that phyllosphere
13 microbiology is greatly more complex than previously understood. Although progress has
14 been made in elucidating the structure and distribution of microbial communities in the
15 phyllosphere, much less is known of the functional consequences of the community or its
16 composition for the fitness of individual plants, the quality and microbiological safety of fresh
17 produce, and wider environmental processes. Microbes reach the phyllosphere by atmospheric
18 deposition from plant and soil sources, but may also colonise plants through the roots, and
19 become transported to aerial parts. The relative importance of these mechanisms remains to
20 be determined. Although microbial establishment and colonisation has long been recognised
21 to be the result of interplay between plant and environmental factors, and the physiological
22 characteristics of microbial colonists, it has now been clearly demonstrated that within plant
23 species, contrasting genotypes can support different microbial communities. This
24 understanding provides opportunities to understand the molecular mechanisms by which
25 plants control microbial populations in the phyllosphere. Such studies could provide methods
18 1 to manipulate phyllosphere communities via plant genotype, providing exciting opportunities Deleted: ¶ Most understanding of phyllosphere microbiology is 2 to manage applied aspects of phyllosphere microbiology, such as the survival of human derived from culture-based studies and although the culture- independent molecular analysis of 3 pathogens or the activity of beneficial microbes. microbial populations in the phyllosphere is still in its infancy, it is clear from recent studies that 4 phyllosphere microbiology is greatly more complex than previously understood. Although 5 progress has been made in elucidating the structure and distribution of microbial 6 Acknowledgements communities in the phyllosphere, much less is known of the functional consequences of the 7 We thank the Department for Environment, Food and Rural Affairs for financial support community or its composition for the fitness of individual plants, the quality and microbiological safety 8 of fresh produce, and wider environmental processes. ¶ There are various developing 9 References technologies which show promise to significantly increase throughput of analysis to provide 10 Adams, P.D. and Kloepper, J.W. (2002) Effect of host genotype on indigenous bacterial a finer resolution of understanding about the diversity and structure of phyllosphere communities and 11 endophytes of cotton (Gossypium hirsutum L.) Plant Soil 240, 181-189. to link diversity with functioning. Culture-independent analysis using phylogenetic specific 12 Andrews, J.H. and Harris, R.F. (2000) The ecology and biogeography of microorganisms of primers represents a powerful method to investigate the dynamics and distribution of 13 plant surfaces. Annu Rev Phytopathol 38, 145-180. specific bacterial groups of interest (Sessitsch et al. 2002; Miyamoto et al. 2004). 14 Arnold, A.E., Maynard, Z., Gilbert, G.S., Coley, P.D. and Kursar, T.A. (2000) Are tropical Additionally multiplex TRFLP, in which several phylogentic groups or functional genes can be 15 fungal endophytes hyperdiverse? Ecol Lett 3, 267-274. analysed at the same time provides an opportunity to improve throughput of samples in 16 Arnold, A.E., Mejia, L.C., Kyllo, D., Rojas, E.I., Maynard, Z., Robbins, N. and Herre, E.A. ... [1] Deleted: There are a number of areas relating to the colonisation 17 (2003) Fungal endophytes limit pathogen damage in a tropical tree. PNAS 100, 15649- of phyllosphere which require more complete understanding. The transmission of 18 15654. microorganisms from roots to aerial parts of plants appears to have been a neglected area of 19 Asins, M.J. (2002) Present and future of quantitative trait locus analysis in plant breeding. research and the importance of this environmentally protected phyllosphere colonisation route 20 Plant Breeding 121, 281-291. needs to be elucidated. This could be particularly important for soils contaminated with human 21 Beattie, G.A. (2002) Leaf surface waxes and the process of leaf colonization by pathogens. Information is needed to characterise the arrangement and dynamics of communities to 22 microorganisms. In Phyllosphere microbiology ed. Lindow, S.E. Hecht-Poinar E.I. and time and space, especially at the landscape scale. In particular. the relative importance of 23 Elliott V.J. pp. 3-26. St. Paul, USA: APS Press. environmental factors, location and plant species in determining the composition and dynamics of 24 Beattie, G.A. and Lindow, S.E. (1995) The secret life of foliar bacterial pathogens on leaves. phyllosphere communities needs to be addressed. Biogeographical approaches to the analysis of 25 Annu Rev. Phytopathol 33, 145-172. microbial communities show potential to elucidate these fundamental relationships (Ramette and Tiedje, 2007).
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32 33 1 Table 1 Percentage frequency of bacterial groups in 16S rRNA gene clone libraries prepared from DNA extracted directly from the phyllospheres 2 of different species. 3 Figures in brackets give total number of sequences obtained Thlapsi Trichilia Trichilia Campomanesia Zea mays1,3 Capsicum Solanum Crocus geosingense1 catigua2 clausenii2 xanthocarpa2 annum1,4 tuberosum1,5 albiflorus1 (76) (109) (153) (166) (30) (39) (137) α-proteobacteria 20.0 10.9 7.8 32.0 30.0 30.8 5.8 15.8 β-proteobacteria 29.0 0.9 1.4 2.4 6.7 17.9 25.5 10.5 γ-proteobacteria 12.0 75.2 63.7 11.4 23.3 25.6 38.6 60.5 Bacteroidetes 17.0 12.9 23.7 20.6 16.7 0.0 2.2 0.0 Cyanobacteria 0.0 0.0 0.0 14.5 0.0 0.0 0.0 2.6 Actinobacteria 4.0 0.0 0.0 1.2 0.0 5.3 8.0 5.3 Firmicutes 12.0 0.0 0.0 13.9 23.3 20.5 19.8 5.3 Acidobacteria 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Reference Idris et al. Lambais et Lambais et Lambais et al. Kadivar and Rasche et Rasche et al. Reiter and 2004 al. 2006 al. 2006 2006 Stapleton 2003 al. 2006b 2006a Sessitsch 2006 4 1 DNA extracted from surface sterilised shoot 5 2DNA extracted from bacterial cells washed from leaf 6 3Field grown, UV and no UV treatments combined 7 4Non chilled and chilled milder Spiral and Ziegenhorn Bello varieties combined
34 1 5 Flowering and senescent Desire and Merkur cultivars combined 2
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Most understanding of phyllosphere microbiology is derived from culture-based studies and although the culture-independent molecular analysis of microbial populations in the phyllosphere is still in its infancy, it is clear from recent studies that phyllosphere microbiology is greatly more complex than previously understood. Although progress has
been made in elucidating the structure and distribution of microbial communities in the
phyllosphere, much less is known of the functional consequences of the community or its
composition for the fitness of individual plants, the quality and microbiological safety of
fresh produce, and wider environmental processes.
There are various developing technologies which show promise to significantly
increase throughput of analysis to provide a finer resolution of understanding about the
diversity and structure of phyllosphere communities and to link diversity with
functioning. Culture-independent analysis using phylogenetic specific primers represents
a powerful method to investigate the dynamics and distribution of specific bacterial
groups of interest (Sessitsch et al. 2002; Miyamoto et al. 2004). Additionally multiplex
TRFLP, in which several phylogentic groups or functional genes can be analysed at the
same time provides an opportunity to improve throughput of samples in a cost effective
manner (Singh et al. 2006). However, these techniques remain time consuming, and future developments will depend on high throughput methods. Phylogenetic microarrays clearly provide a way forward, allowing the presence and amount of thousands of microorganisms to be determined simultaneously, and could also be used to detect novel members of phylogenetic groups. Similarly, functional gene arrays provide a means of characterising activity of the phyllosphere community, and when used with phylogenetic
microarrays, for linking community structure to function (Sessitsch et al., 2006).
Although many studies have demonstrated that plant genotype is an important factor
determining the structure of phyllosphere microbial communities, the mechanisms
controlling these interactions remain to be elucidated. Various plant science resources are
available which show potential for examining plant genotype-phyllosphere microbiology
interactions. In particular recombinant inbred mapping populations (Asins, 2002) have the potential to identify plant genes controlling leaf microbiology. Such studies could provide methods to manipulate phyllosphere communities via plant genotype, providing opportunities to manage applied aspects of phyllosphere microbiology, such as the survival of human pathogens or the activity of beneficial microbes.